LIU Li,JI Hui,PENG Lin,et al.The establishment and optimization of the drug metabolic model of adult chicken primary hepatocytes[J].Journal of Nanjing Agricultural University,2015,38(1):127-133.[doi:10.7685/j.issn.1000-2030.2015.01.019]





The establishment and optimization of the drug metabolic model of adult chicken primary hepatocytes
刘丽 季辉 彭麟 阮祥春 吉利伟 江善祥
南京农业大学动物医学院, 江苏 南京 210095
LIU Li JI Hui PENG Lin RUAN Xiangchun JI Liwei JIANG Shanxiang
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
adult chickenin vitro perfusionchicken hepatocytesdrug metabolic modeloptimization
[目的]建立成年鸡原代肝细胞的药物代谢模型并对其进行优化.[方法]以改进的二步灌流法分离8~12周龄的雄性黄羽鸡肝细胞,比较在不同细胞基础培养液条件下鸡肝细胞体外培养的情况,利用倒置显微镜观察肝细胞的形态,利用MTT检测法绘制生长曲线,测定细胞培养上清液中乳酸脱氢酶(LDH)活性,用qPCR和Western blot测定肝细胞中CYP450酶亚型CYP1A4、CYP1A5CYP3A37 mRNA相对表达水平和3种酶蛋白的表达量.[结果]离体灌流操作简便,获得的细胞存活率高达90%以上;用含0.5 mg·L-1牛胰岛素、10 g·L-1双抗(100 U·mL-1青霉素和链霉素)和体积分数为10%胎牛血清的DMEM基础培养液体外培养鸡肝细胞,贴壁培养时可见到肝细胞较为明显的分化过程,贴壁后呈上皮细胞样生长,多角形,胞浆内容物均匀丰富,细胞核清晰透亮,少数有双核,细胞生长状态良好.生长曲线和LDH活性测定结果显示:培养3~5 d时细胞状态稳定,并且在肝原代细胞培养的8 d内,CYP1A4、CYP1A5CYP3A37 mRNA相对表达水平和蛋白的表达量没有显著差异.[结论]以改进的二步灌流法分离鸡肝细胞操作简便,分离的鸡肝原代细胞存活率高,用含0.5 mg·L-1胰岛素、100 U·mL-1青霉素、100 μg·mL-1链霉素和10%胎牛血清的DMEM基础培养液体外培养鸡肝原代细胞效果最好,培养3~5 d是进行体外药物代谢等试验的最佳时机.
[Objectives]To establish the drug metabolic model in vitro, the primary hepatocytes culture methods and the drug metabolic model were optimized. [Methods]The chicken primary hepatocytes were isolated by modified two-step in situ collagenase perfusion from 8-10 week-old yellow-feathered male broilers. Meanwhile, effects of different mediums on the morphology and growth of the cells were investigated. The culture condition was optimized by comparing the effects between DMEM medium, 1640 medium, William’s E medium and Leibovitz’s L-15(L-15)medium. The morphology of the cells was observed under the inverted microscope and the growth curve was drawn by MTT assay. The lactate dehydrogenase(LDH)concentration in supernatant was determined with a LDH test kit. And the relative expression of mRNA about CYP1A4, CYP1A5 and CYP3A37 was quantitated by the qPCR method, while protein relative expression about CYP1A4, CYP1A5 and CYP3A37 was quantitated by Western blot. [Results]The isolated method of the chicken primary hepatocytes by modified two-step in situ collagenase perfusion in vitro is simple. The well-dispersed and pure hepatocytes with high viability(>90%)were obtained. By comparing the effects of the four mediums, the adherence of the chicken primary hepatocytes were best by using William’s E medium, followed by DMEM medium and L-15 medium, then 1640 medium slightly worse. But the chicken primary hepatocytes maintained a long stable period on the morphology and growth in the long process by using DMEM medium, long-term training 2-3 weeks, and compared with William’s E medium, DMEM medium is more economical, so the chicken primary hepatocytes were cultured by using DMEM medium supplemented with 0.5 mg·L-1 insulin, 1% streptomycin and 10% fetal bovine serum foundation. The cells attached on the surface of petri dishes with 4 h after inoculation(5×105 mL-1). The morphology of chicken hepatocytes which was observed under the inverted microscope was effectively maintained in the culture medium. Under this condition, the cells were cultured for 8 d keeping their typical parenchymal morphology, with characteristic polygonal shape and formation of bile canaliculi. The growth curve result showed the growth state of the primary culture chicken hepatocytes(MTT assay)cultured in DMEM medium was downward trend, but the decline trend was not obvious between 3-5 d. And the LDH concentration results showed that activity of the hepatocytes was stable from 3-5 d after inoculation. And during the cells cultured in the culture medium for eight days, there were no significantly differences about the relative expression of mRNA and protein about CYP1A4, CYP1A5 and CYP3A37 in cells. [Conclusions]The chicken primary hepatocytes were isolated by modified two-step in situ collagenase perfusion in vitro which is simple. The well-dispersed and pure hepatocytes with high viability were obtained. The chicken hepatocytes cultured in vitro were more economic and better by using DMEM medium supplemented with 0.5 mg·L-1 insulin, 10 g·L-1streptomycin and 10% fetal bovine serum foundation. The drug metabolism test in vitro was suitable between 3-5 d, and the foundation for follow-up pilot study on drug metabolism research in vitro was thus established.


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更新日期/Last Update: 1900-01-01